TISSUE ENGINEERING: Part C Volume 17, Number 2, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2010.0200

Human Primary Articular , -Like Cells, and Dedifferentiated Chondrocytes: Differences in Gene, MicroRNA, and Expression and Phenotype

Tommy A. Karlsen, M.Sc., Aboulghassem Shahdadfar, Ph.D., and Jan E. Brinchmann, M.D., Ph.D.

In this study we have isolated human primary uncultured articular chondrocytes. When these cells are allowed to proliferate within their own extracellular (ECM), they begin to produce hyaline ECM molecules similar to embryological chondroblasts. These cells are called -like cells. Upon continued culture these cells spread onto the plastic surface and dedifferentiate. We have characterized these three stages of chondral cells by gene expression and expression of microRNAs (miRNAs) and . Gene expression was quantified by real- time reverse transcriptase (RT) polymerase chain reaction, miRNA expression by miRNA arrays, and protein synthesis by extra- and intracellular flow cytometry. Many of the genes, miRNAs, and proteins were differentially expressed in the different stages of chondral cells. In the context of cellular therapy, expression of some genes is a cause for concern. The best source of cells for treatment of lesions of hyaline has not yet been identified. Adult chondroblast-like cells may be strong candidates. Profound understanding of how expression of genes and synthesis of proteins are regulated in these cells, for instance, by miRNAs, may reveal new strategies for improving their synthesis of hyaline ECM. This insight is important to be able to use these cells in the clinic.

Introduction the features of embryological chondroblasts, they are called chondroblast-like cells (CBLC). utologous implantation (ACI) is now Although the use of CBLC in clinical transplantation Astandard therapy in many centers for patients with focal might well improve on the clinical results obtained by using lesions of hyaline articular cartilage.1,2 ACI requires the re- dedifferentiated chondrocytes, this has not yet been dem- moval of a small piece of articular cartilage, followed by onstrated clinically. We are presently conducting clinical ex vivo expansion of its content of articular chondrocytes trials using these cells. However, to further improve (AC) to obtain large number of cells for implantation. Ex vivo therapy strategies it is important to fully understand the expansion of AC in monolayer cultures invariably leads to biology of the cell populations used. This includes quantifi- dedifferentiation of the chondrocytes.3,4 This means that the cation of the genes expressed, the proteins they encode, and AC revert to a less specialized cell type, where synthesis of the understanding the mechanisms that regulate gene expres- -associated proteins type II (COL2) sion. MicroRNAs (miRNAs) are a recently discovered class and (ACAN) is replaced by synthesis of COL1 and of molecules that affect both mRNA expression and protein (VCAN), respectively.5 These synthesis. miRNAs are small single-stranded RNA molecules (ECM) components are typical of fibrocartilage.6,7 Trans- (*21–22 nt). miRNAs are derived from long primary tran- plantation of dedifferentiated chondrocytes frequently leads scripts (pri-miRNA, 2 kb in length) that are mainly tran- to the formation of fibrocartilage, or a mixture of fibrocartilage scribed by the RNA polymerase II. In the nucleus the and hyaline cartilage within the lesion.8,9 It is believed that this pri-miRNAs are processed by the RNase Drosha to create repair has inferior functionality and durability, and that short stem-loop molecules called pre-miRNAs (*70 nt) that an end result may be arthrosis in that .10 Most likely, a cell are exported to the cytoplasm. In the cytoplasm the pre- population more similar to the AC found in vivo is needed for miRNAs are further processed by the cytoplasmic RNase implantation to improve the outcome of ACI. Recently, we Dicer into a 21–22 nt miRNA duplex. The miRNA duplex is established a new culture system where AC were cultured in then assembled into the RNA-induced silencing complex their own ECM.11 At day 7–10 of culture the cells maintained a where one of the strands (mature miRNA) base-pair with rounded shape and expressed genes for several hyaline ECM complementary mRNA transcripts.12,13 It is thought that molecules while still proliferating. As these cells have many of perfect complementary leads to degradation of the mRNA,

Institute of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway.

219 220 KARLSEN ET AL. whereas imperfect complementary results in translational systems, Abingdon, United Kingdom). Relative quantification inhibition.14,15 It is generally accepted that the main function was performed using the 7300 Real-Time reverse transcriptase of miRNA is to knock down gene activity, although it has (RT) PCR system (Applied Biosystems) and TaqMan Gene been shown to activate transcription also.16 One-third of all Expression assays following protocols from the manufacturer human genes have been estimated to be regulated by miR- (Applied Biosystems). TaqMan assay numbers are listed in NA.17 As expression of individual miRNA relatively easily Table 1. All samples were run in technical triplicates contain- may be either increased or decreased by introducing miRNA ing 1.0 mL cDNA in a total volume of 25 mL. The thermocycling precursor molecules or antagonists,18 insight into expression parameters were 958C for 10 min followed by 40 cycles of 958C of miRNA in the different types of chondral cells may in- for 15 s and 608C for 1 min. Validation of 32 different endog- troduce a new strategy for enhancing expression of genes enous controls was performed using the TaqMan Human expressing ECM molecules or decreasing expression of genes Endogenous Control Plate following protocols from the and proteins associated with dedifferentiation. manufacturer (Applied Biosystems). Subsequently, all sam- The objective of this study was to investigate the three cell ples were scaled relative to expression level of CDKN1A, populations: primary uncultured chondrocytes, CBLC, and which was found to be one of the most suitable endogenous dedifferentiated chondrocytes with respect to their expres- controls in our experimental settings. sion of a large number of genes, proteins, and miRNA related to . The results add to our under- miRNA array and real-time RT polymerase chain standing of the biology of in vitro chondrogenesis, and may reaction validation also introduce new strategies for manipulation of chon- Total RNA enriched with small RNA molecules (including drocytes used in clinical protocols. miRNA) was extracted from the cells using the Trizol (In- vitrogen, Carlsbad, CA) procedure with the following modifi- Material and Methods cations: 1 mL of isopropanol was included in the precipitation Chemicals step and the samples were kept at 808C for minimum 30 min. After centrifugation the RNA pellet was dissolved in Trizol and All chemicals were purchased from Sigma-Aldrich (St. column-purified using reagents and following protocols from Louis, MO) unless otherwise stated. the miRNeasy kit (Qiagen, Hilden, Germany). After DNase treatment (Ambion), the total RNA was Isolation and culture of the cells quantified by spectrophotometry (Nanodrop). miRNA pro- Articular cartilage was obtained from the femoral surface of filing was obtained through the miRNA microarray service, three obtained from legs amputated for reasons other including in depth analysis, from LC Sciences (Houston, TX, than cartilage disease. The donors were 84, 79, and 76 years of www.lcsciences.com). Analysis of variance ( p < 0.01) was age, and all provided written informed consent. The study was used for clustering to make a heat map. The arrays were approved by the Regional Committee for Medical Research based on the Sanger miRBase Release 13.0 and contained 875 Ethics, Southern Norway, Section A. The cells was isolated as unique probes. For real-time reverse transcriptase (RT) previously described.11 Briefly, all the articular cartilage with polymerase chain reaction (PCR) validation, the miRNAs normal and healthy appearance was harvested and cut into were reverse transcribed to cDNA using primers included in small pieces. Only for one donor (donor 2) there were minor the TaqMan miRNA assays (Table 1) and reagents from the areas of the cartilage with abnormal appearance. This patient TaqMan miRNA RT kit following protocols from the man- had no previous history of in the . The car- ufacturer (Applied Biosystems). Relative quantification was tilage pieces were digested with Collagenase type XI (1.2 mg/ performed using the same parameters as described above for mL) at 378C for 90–120 min. The ensuing structures, called the real-time RT PCR analysis. CDKN1A was used as en- chondrocytes in autologous ECM, were washed several times dogenous control. The prediction tools PicTar (http://pictar. and resuspended in culture medium consisting of Dulbecco’s mdc-berlin.de/), TargetScan (www.targetscan.org/), miRDB modified Eagle’s medium/F12 supplemented with 10% fetal (http://mirdb.org/), Diana Micro-T (http://diana.cslab.ece. bovine serum, 50 mg/mL ascorbic acid, 100 units/mL penicil- ntua.gr/), and miRNA viewer (http://cbio.mskcc.org/ lin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B. mirnaviewer/) were used to search for possible miRNA targets. The culture medium was changed every 3–4 days. After the first passage (10–12 days) amphotericin B was removed. At Flow cytometric analysis 60% confluence, cells were detached with trypsin-ethylene- For flow cytometric analysis uncultured cells were di- diaminetetraacetic acid and seeded into new culture flasks. gested with collagenase for 3–4 h and passed first through 100 mm and then 40 mm pore size filters. The following di- Gene expression analysis using real-time rectly conjugated monoclonal (Mab) were used reverse transcriptase polymerase chain reaction for cell surface marker staining: CD49b (integrin a2)/ Total RNA for gene expression analysis was isolated using PE, CD49f (a6)/PE, CD90/PE, CD104 (b4)/FITC, CD106/ the RiboPure kit following protocols from the manfacturer FITC, CD146/FITC (AbD Serotec, Kidlington, United King- (Ambion, Austin, TX). After DNase treatment (Ambion), dom), CD34/FITC, 45/FITC, CD49a (a1)/FITC, CD49c (a3)/ RNA was quantified by spectrophotometry (Nanodrop, PE, CD49e (a5)/PE, CD29 (b1)/APC, CD44/PE, CD73/PE, Wilmington, DE). For each reaction 200 ng total RNA was CD117/PE (BD Biosciences, San Diego, CA), CD51 (aV)/ reverse transcribed into cDNA (total volume of 20 mL) by PE (Chemicon Internationals, Temecula, CA), CD19/ using the High Capacity cDNA Reverse Transcription kit APC, CD105/APC, HLA-DR/APC (Diatec, Oslo, Norway), following protocols from the manufacturer (Applied Bio- CD144/PE, Integrin a5/FITC (eBioscience, San Diego, CA), GENE, PROTEIN, AND MICRORNA EXPRESSION IN CHONDRAL CELLS 221

Table 1. TaqMan Gene and MicroRNA Expression Assays

Gene symbol Gene name TaqMan assay no.

ACAN Aggrecan Hs00202971_m1 ALPL Alkaline phosphatase Hs00758162_m1 ADAMTS4 ADAM metallopeptidase with thrombospondin type 1 motif, 4 Hs00192708_m1 ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5 Hs00199841_m1 BGLAP gamma-carboxyglutamate protein (Osteocalcin) Hs01587813_g1 CDKN1A Cyclin-dependent kinase inhibitor 1A Hs00355782_m1 COL1A1 Hs00164004_m1 COL2A1 Type II collagen Hs00264051_m1 COL9A1 Type IX collagen Hs00156680_m1 COL10A1 Type X collagen Hs00166657_m1 COL11A1 Type XI collagen Hs00266273_m1 COMP Cartilage oligomeric matrix protein Hs00164359_m1 DCN Decorin Hs00754870_s1 HAS2 Hyaluronan synthase 2 Hs00193435_m1 ITGA2 Integrin, alpha 2 Hs00158148_m1 ITGA6 Integrin, alpha 6 Hs00173952_m1 ITGA10 Integrin, alpha 10 Hs00174623_m1 MMP1 Matrix metallopeptidase 1 Hs00899658_m1 MMP3 Matrix metallopeptidase 3 Hs00869308_m1 MMP13 Matrix metallopeptidase 13 Hs00233992_m1 OMD Osteomodulin Hs00192325_m1 RUNX2 Runt-related 2 Hs00231692_m1 SOX5 SRY (sex determining region Y)-box 5 Hs00374709_m1 SOX6 SRY (sex determining region Y)-box 6 Hs00264525_m1 SOX9 SRY (sex determining region Y)-box 9 Hs00165814_m1 VCAN Versican Hs01007941_m1 MicroRNA miR-140-3p microRNA 140 002234 miR-145 microRNA 145 002278 miR-222 microRNA 222 002276 miR-451 microRNA 451 001141

CD49d (a4)/FITC (Immunotec, Quebec, Canada), CD133/ Results FITC (Miltenyi Biotec, Gladbach, Germany), and CD14/ During the first 7–10 days of culture, most of the AC FITC (Sigma-Aldrich). The cells were coated with directly were proliferating inside the chondrocytes in autologous conjugated Mab at room temperature for 20 min, washed ECM (three-dimensional culture) as previously described.11 with phosphate-buffered saline, and fixed in 1% paraform- These cells have previously been shown to produce hyaline aldehyde. Intracellular staining was performed following matrix molecules,11 and are here called CBLC.19 After 10– the protocols from the Cytofix/Cytoperm Fixation/ 13 days increasing numbers of cells adhered to the surface Permeabilization kit (BD Biosciences). The following uncon- of the culture flasks, and after 17–19 days all the cells were jugated antibodies were used: Aggrecan (Biosource, Nivelles, proliferating in adherent monolayer cultures. AC from Belgium), Type I Collagen, Type II Collagen (MP biomedi- donor 2 spread onto the plastic surface 2–3 days earlier cals, Aurora, OH), Versican (R&D Systems, Abingdon, than cells from donors 1 and 3. At day 28 cells from all United Kingdom), Integrin a7a, Integrin a7b (gift from donors had been in monolayer with fibroblastic morphol- Dr. Tarone), Integrin a10 (gift from Hansa Medical AB), and ogy for at least 10 days and were termed dedifferentiated Integrin a11 (gift from Dr. Donald Gullberg). Cells were first chondrocytes. incubated with unconjugated primary antibodies against the molecule of interest for 20 min, washed, and then incubated with a fluorochrome-conjugated species and isotype-specific Endogenous control for real-time RT PCR secondary . Irrelevant Mabs were used as controls As recommended,20 several endogenous controls were for all fluorochromes. Cells were analyzed using a FACS- analyzed before the gene and miRNA expression experi- Calibur flow cytometer (Becton, Dickinson, San Jose, CA; ments were performed. Of the 32 possible endogenous con- www.bd.com). Median fluorescence index was calculated as trols on the TaqMan Human Endogenous Control Plate follows: median fluorescence intensity of target molecule CDKN1A had one of the most stable gene expression in our median fluorescence intensity of isotype control. experimental settings. Several of the standard housekeeping genes such as GAPDH, b-actin, and 18s RNA were expressed Statistical analysis at much lower levels in primary uncultured chondrocytes The in-depth analysis of miRNA from LC Sciences than in proliferating cells (Supplementary Fig. 1; Supple- (www.lcsciences.com) included analysis of variance ( p < 0.01), mentary Data are available online at www.liebertonline which was used for clustering to make a heat map. .com/ten).

FIG. 1. Relative quantification of gene expression by real-time RT PCR. Real-time reverse transcriptase (RT) PCR analysis of relevant genes expressed during the three stages of in vitro culture. Stage 1: uncultured primary AC (day 0). Stage 2: adult chondroblast-like cells (day 7 and 14). Stage 3: dedifferentiated chondrocytes (day 28). Gene expression levels were scaled relative to CDKN1A expression levels. Data are shown as mean standard deviation for technical triplicates in each of three donors. Note log scale for the different Y axes. PCR, polymerase chain reaction.

222 GENE, PROTEIN, AND MICRORNA EXPRESSION IN CHONDRAL CELLS 223

Changes in gene expression during in vitro culture Real-time RT PCR was used to determine changes in gene expression of a large number of genes (Fig. 1). The AC went through three stages during the 28 days of in vitro culture: (1) primary, uncultured nonproliferating AC (day 0); (2) the chondroblast-like stage (day 7–14); and (3) the dedifferenti- ation stage at the end of the culture. These stages were characterized by differential expression of genes encoding many of the and relevant transcription factors (TFs) (Fig. 1). COL2A1, COL9A1, and COL11A1 levels were high or moderate in uncultured cells. Expression increased 10–300- fold during stage 2, and then fell to low levels in stage 3, particularly for COL9A1 and COL2A1. COL10A1 levels were moderate in uncultured cells, were slightly downregulated during stage 2, and fell to undetectable or very low levels in stage 3. Moderate levels of COL1A1 were detected in un- cultured AC, and expression increased gradually to very high levels in dedifferentiated cells. The best known regulators of expression of COL2A1 are the TFs SOX9, 5, and 6.21 High levels of SOX9 where de- tected in uncultured AC at stage 1. Surprisingly, SOX9 levels did not increase in parallel with COL2A1 levels, but instead weakly decreased during stage 2 and dramatically decreased during stage 3. In contrast, SOX5 and SOX6 levels were low/ moderate in uncultured cells and then changed in parallel with COL2A1 during stage 2 and 3. Gene expression of other molecules of importance for hyaline matrix was also inves- tigated. We saw gradual increases in VCAN and HAS2 and gradual decrease in COMP in the course of cell culture, with minor changes observed for ACAN and DCN. ITGA2 de- creased from day 7, whereas ITGA6 and ITGA10 increased at the beginning of stage 2 (day 7) and then decreased. Figure 1 also shows gene expression of several matrix degrading en- FIG. 2. Heat map of miRNA expressed in the three differ- zymes. Interestingly, MMP3 was detected at high levels in ent stages of culture. Clustered presentation of miRNA uncultured cells. MMP1, MMP3, and MMP13 were greatly expressed differentially ( p < 0.01, analysis of vasriance) be- increased at day 7, followed by a decrease in expression to tween the three stages of in vitro culture. Red and green color that observed at day 0 or lower. The gene encoding the ag- indicates high and low expression, respectively. D1 ¼ Donor grecan degrading enzyme ADAMTS4 was not drastically 1, D2 ¼ Donor 2, and D3 ¼ Donor 3. miRNA, microRNA. changed, whereas ADAMTS5 slightly increased in dediffer- entiated AC. Some bone-related genes were also examined. miR-140-3p, miR-222, and miR-145 was very similar to that ALPL and RUNX2 levels increased after stage 1. BGLAP obtained from miRNA arrays (Fig. 3A–D). (Osteocalcin) and OMD expression was not consistent be- tween the donors. Changes in cell surface marker and ECM protein expression during in vitro culture miRNA profiling Cells use adhesion molecules, such as integrins, to attach miRNAs are a class of recently discovered small RNA to their ECM ligands in vivo and to the plastic surface of the molecules that may regulate important cellular functions culture flasks during in vitro expansion. Therefore, we also during in vitro culture of AC. On the basis of our gene ex- analyzed expression of different integrin subunits and other pression results, we chose day 0 (stage 1, uncultured cells), cell surface markers using flow cytometry. All integrin sub- day 7 (stage 2), and day 28 (stage 3) for global miRNA anal- units except for a6, a7a, a7b, and a11 were detected on un- ysis. Hierarchical clustering of differentially ( p < 0.01) ex- cultured cells. a2, a3, b3, and b4 were only detected in one pressed miRNA revealed four different clusters (Fig. 2). One donor (low levels). After 28 days of in vitro culture expres- cluster consisted of miR-451, which was only upregulated in sion of most integrin subunits increased (Fig. 4A). CD44, stage 1. Four miRNA, including miR-140-3p, were upregu- CD73, CD90, CD105 (markers expressed by mesenchymal lated in COL2A1 producing cells (stage 1 and 2). Another stem cells [MSC]), and CD106 were also expressed by the cluster consisted of five miRNA, including miR-221 and miR- uncultured AC. Again expression increased at day 28. To 222, that were upregulated in proliferating cells (stage 2 and ensure that there was no contamination of haematopoietic 3). The last cluster consisted of 11 miRNA, including miR-143 cells in our cultures, we also analyzed expression of CD14, and miR-145, upregulated only in dedifferentiated cells (stage CD19, CD34, and CD45. These markers were not found on 3). One representative miRNA from each cluster was vali- any of the cells. Also CD117, CD133, CD144, and HLA-DR dated using real-time RT PCR. Expression pattern of miR-451, were not detected. 224 KARLSEN ET AL.

FIG. 3. Validation of miRNA expression by real- time RT PCR. One miRNA from each of the four clusters, i.e., miR-451 (A), miR-140-3p (B), miR-222 (C) and miR-145 (D), was validated with real- time RT PCR. miRNA levels were scaled relative to CDKN1A expression levels. Data are shown as mean standard deviation for technical triplicates in each of three do- nors. Note linear scale for the

different Y axes.

Intracellular levels of COL1 were barely detected in two of able to produce perfect hyaline ECM. The only cells known the donors in the uncultured cells. However, higher levels to make hyaline ECM are embryonic chondroblasts19 and were found at days 14 and 28 in all three donors (Fig. 4B). adult chondrocytes. Thus, our therapeutic cells should per- COL2 was detected at all time points, with slightly higher haps resemble embryonic chondroblasts. levels at day 14. ACAN was expressed at moderate levels Genes encoding several collagens changed in the course of and was not differentially expressed during culture. VCAN cell culture according to three different patterns. The first was not expressed in the uncultured cells, whereas it was pattern was followed by COL2A1, COL9A1, and COL11A1 present in all three donors at days 14 and 28. levels, which increased during stage 2 and then decreased during stage 3. In hyaline cartilage the collagen fibrils consist Discussion of bundles of COL2 with small amounts of COL11. COL9 Presumably, the best therapeutic strategy for lesions of binds in a regular pattern to the surface of the COL2/COL11 articular cartilage will be based on the use of cells that are fibrils and is thought to mediate interaction with other col- GENE, PROTEIN, AND MICRORNA EXPRESSION IN CHONDRAL CELLS 225

small amounts of COL1 in the uncultured cells. This may suggest a blockage of translation in these cells. For day 14 and 28 the intracellular COL1 matched the COL1A1 levels. This may explain why dedifferentiated chondrocytes, when used for clinical transplantation, frequently produce fibrocartilage dominated by COL1.9 Expression of SOX5 and SOX6 increased in stage 2 cells, whereas SOX9 was already highly expressed in the uncul- tured cells. During embryogenesis, SOX9 is expressed in prechondrocytes, before SOX5 and SOX6. Low levels of COL2A1 are also expressed in these cells.19 In embryological chondroblasts all these genes are expressed, and COL2 pro- tein and hyaline cartilage is being synthesized. Thus, similar to embryological chondrogenesis, the upregulation of SOX5 and SOX6 on a background of SOX9 availability may be important effectors of chondroblast differentiation.19 ACAN and VCAN levels were mirrored by protein ex- pression for both molecules. Although the results for VCAN were entirely as expected, ACAN levels are frequently found to be reduced in dedifferentiated chondrocytes.30 One pos- sible explanation for the persistently high ACAN levels in these cells could be that day 28 cells may not have entirely completed the dedifferentiation process. FIG. 4. Cell surface marker and intracellular ECM staining In stage 2, expression of MMP1, 3, and 13 dramatically in uncultured and dedfferentiated AC. (A) Integrin expres- increased at day 7 before returning to baseline expression or sion on day 0 (uncultured chondrocytes) and day 28 lower. In embryological chondrogenesis MMPs are upregu- (dedifferentiatied chondrocytes) of culture. (B) Cell surface lated to soften the ECM,31 thus, providing space for the cells marker and intracellular ECM molecule expression at days 0, 14, and 28. The bars represent MFI for the three donors, with to migrate and establish cell–cell contact important for con- error bars representing the range. *Not detected. ECM, ex- densation, the first step in chondrogenesis. Interestingly, tracellular matrix; MFI, median values for fluorescence in- several MMPs, including MMP3 and MMP13, were found to 32 tensity. be expressed during chondrogenic differentiation of MSC. MMP13 is also known to be a marker for hypertrophy during embryological chondrogenesis.19,33 Further investigations lagen fibrils and matrix proteins.22 Given this relationship are required to determine the significance of these MMPs in in vivo, and the fact that COL2A1, COL9A1, and COL11A1 are our culture system. The increase of ALPL and RUNX2 is a activated by SOX9,23–25 it is perhaps not surprising that they potential concern as they are markers of osteogenesis, and seem to be co-regulated. For COL2, we observed the same also of chondroblast hypertrophy during embryogenesis.19 expression pattern at the protein level, though at low levels These genes were expressed at low levels in adult CBLC. and with relatively small differences between the cell pop- Importantly, no evidence of bone formation or unexpected ulations. The low level of COL2 in the uncultured cells may cell death has so far been observed with the clinical use of be explained by the fact that collagen in cartilage has a half- these cells. Still, expression of these genes requires attention life of >100 years.26 The requirement for new COL2 in adult in further studies. cartilage would thus be expected to be low. We have pre- We found a number of changes in expression of integrins viously shown that CBLC produce hyaline matrix containing and other cell surface markers when AC were established in COL2 fibrils.11 The relatively small increase in intracellular culture. These results correspond almost exactly to observa- COL2 levels between uncultured chondrocytes and CBLC tions made by Woods et al.34 At day 28 of culture expression may reflect rapid transport of COL2 molecules to the extra- of most subunits had increased. This suggests that ACs use cellular space. different set of integrins to adhere to ECM and the culture A second pattern was followed by COL10A, which re- flasks. mained largely unchanged in the CBLC, and then dropped to We also show here that many miRNAs were differently very low or undetectable levels in dedifferentiated cells. expressed in cells of the different stages. MiR-451 was the Expression of COL10A1 is a marker for hypertrophy during only miRNA that was highly expressed only in the primary embryogenesis.19,27 However, COL10 is found in both the uncultured AC. This miRNA has been shown to reduce superficial and near the tidemark zone in normal human proliferation in cells35,36 and may perhaps have a role articular cartilage.28 Thus, COL10A1 expression in these cells as inhibitor of proliferation also in AC in vivo. Of the miRNA may possibly reflect physiological requirements for COL10, highly expressed in both stages 1 and 2, miR-140-3p is of and not the ominous onset of hypertrophy. particular interest as it was the miRNA with the highest Following a third pattern, COL1A1 gradually increased expression in our dataset (the complementary sequence miR- from moderate levels to very high levels in dedifferentiated 140-5p showed the same expression pattern and was signif- cells. In vivo COL1 is known to be a part of the ECM in the icant with p < 0.05; data not shown). miR-140 has been superficial zone, but is not normally synthesized by AC in shown to be cartilage specific in mouse embryos where it other zones.29 By intracellular flow cytometry we found very targets histone deacetylace 4 (HDAC4).37 Recently, miR-140 226 KARLSEN ET AL. was also shown to regulate homeostasis and development of References cartilage.38 This group of miRNA was downregulated in 1. Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, dedifferentiated cells only, suggesting that they may inhibit O., and Peterson, L. Treatment of deep cartilage defects in molecules participating in the dedifferentiation process. Four the knee with autologous chondrocyte transplantation. N of the five miRNAs that were upregulated in proliferating Engl J Med 331, 889, 1994. cells (stages 2 and 3) (miR-17, 31, 221, and 222) have been 2. Brittberg, M. Autologous chondrocyte implantation— 39–43 shown to induce proliferation in several other cell types, technique and long-term follow-up. Injury 39 Suppl 1, S40, again suggesting miRNA participation in the regulation of 2008. cell cycle in these cells. Of the 11 miRNAs that were upre- 3. Benya, P.D., and Shaffer, J.D. Dedifferentiated chondrocytes gulated in dedifferentiated AC only, miR-145, 132, 138, and reexpress the differentiated collagen phenotype when cul- let-7e are interesting. Several miRNA prediction tools predict tured in gels. Cell 30, 215, 1982. miR-145 to target SOX9 and COL2A1. SOX5 is predicted to 4. von der Mark, K., Gauss, V., von der Mark, H., and Muller, be a target of miR-132, SOX6 and COL11A2 are possible P. Relationship between cell shape and type of collagen targets of miR-138, and let-7e may have P4HA2 (prolyl 4- synthesised as chondrocytes lose their cartilage phenotype hydroxylase, a key enzyme in collagen synthesis) as a target. in culture. 267, 531, 1977. This may, potentially, contribute to the dedifferentiation of 5. Binette, F., McQuaid, D.P., Haudenschild, D.R., Yaeger, P.C., the cells. In contrast to our observations, Dunn et al. found McPherson, J.M., and Tubo, R. Expression of a stable artic- decreased miR-145, 221, and 222 levels in monolayer cells ular cartilage phenotype without evidence of hypertrophy compared to native cartilage.44 As our observations for miR- by adult human articular chondrocytes in vitro. J Orthop Res 145 and 222 were made in global arrays and confirmed by 16, 207, 1998. real-time quantitative PCR, we believe that our results are 6. Eyre, D.R., and Muir, H. Quantitative analysis of types I and II collagens in human intervertebral discs at various ages. correct for our experimental system. The discrepancy may be Biochim Biophys Acta 492, 29, 1977. explained by the fact that Dunn et al. compared bovine AC 7. Melrose, J., Smith, S.M., Appleyard, R.C., and Little, C.B. derived from distinct zones and from anatomically distinct Aggrecan, versican and type VI collagen are components of regions in the , which introduces both species and annular translamellar crossbridges in the . possibly anatomical reasons for the differences in miRNA 45,46 47 Eur Spine J 17, 314, 2008. expression. MiR-27 and miR-675 have also been shown 8. Grigolo, B., Roseti, L., De Franceschi, L., Piacentini, A., to be involved in cartilage biology. However, these miRNAs Cattini, L., Manfredini, M., Faccini, R., and Facchini, A. were not differently expressed in the three different cell Molecular and immunohistological characterization of hu- populations studied here. man cartilage two years following autologous cell trans-

In conclusion, we demonstrate that CBLC express genes plantation. J Bone Joint Surg Am 87, 46, 2005. and synthesize proteins typical of hyaline ECM. We describe 9. Knutsen, G., Drogset, J.O., Engebretsen, L., Grontvedt, T., differences in gene and miRNA expression between CBLC Isaksen, V., Ludvigsen, T.C., Roberts, S., Solheim, E., Strand, and uncultured chondrocytes as well as dedifferentiated T., and Johansen, O. A randomized trial comparing autolo- chondrocytes. We also describe the differential expression of gous chondrocyte implantation with microfracture. Findings miRNAs in the three different cell types. As miRNAs regu- at five years. J Bone Joint Surg Am 89, 2105, 2007. late gene as well as protein expression, this insight may 10. Shelbourne, K.D., Jari, S., and Gray, T. Outcome of untreated improve our understanding of chondrocyte biology and also traumatic articular cartilage defects of the knee: a natural introduce new therapeutic targets for cell-based treatment history study. J Bone Joint Surg Am 85A Suppl 2, 8, 2003. strategies for lesions of hyaline cartilage. Whether CBLC will 11. Shahdadfar, A., Loken, S., Dahl, J.A., Tunheim, S.H., Collas, produce better hyaline cartilage than dedifferentiated chon- P., Reinholt, F.P., Engebretsen, L., and Brinchmann, J.E. drocytes or MSCs need to be tested in formal assays for Persistence of collagen type II synthesis and secretion in rapidly proliferating human articular chondrocytes in vitro. in vitro chondrogenesis48 and, eventually, by clinical trials. Tissue Eng Part A 14, 1999, 2008. Acknowledgment 12. Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. Asymmetry in the assembly of the RNAi en- This work was supported by Storforsk and Stamceller zyme complex. Cell 115, 199, 2003. grants from the Research Council of Norway and Gidske og 13. Kim, V.N. MicroRNA biogenesis: coordinated cropping and Peter Jacob Sørensens Foundation for the Promotion of Sci- dicing. Nat Rev Mol Cell Biol 6, 376, 2005. ence. We acknowledge Dr.Tarone (Molecular Biotechnology 14. Hutvagner, G., and Zamore, P.D. A microRNA in a multiple- Center, University of Torino, Italy) for kindly providing turnover RNAi enzyme complex. Science 297, 2056, 2002. antibody raised against integrin a7a and a7b, and Hansa 15. Lee, R.C., Feinbaum, R.L., and Ambros, V. The C. elegans Medical AB (Lund, Sweden) for kindly providing antibody heterochronic gene lin-4 encodes small RNAs with antisense specific for integrin a10 and Dr. Gullberg (Department of complementarity to lin-14. Cell 75, 843, 1993. Biomedicine, University of Bergen, Norway) for kindly 16. Vasudevan, S., Tong, Y., and Steitz, J.A. Switching from providing antibody raised against integrin a11. repression to activation: microRNAs can up-regulate trans- lation. Science 318, 1931, 2007. Disclosure Statement 17. Lewis, B.P., Burge, C.B., and Bartel, D.P. Conserved pairing, often flanked by adenosines, indicates that thousands This work has not received financial support or other of human genes are microRNA targets. Cell 120, 15, 2005. benefits from commercial sources, and none of the authors 18. Huang, J., Zhao, L., Xing, L., and Chen, D. MicroRNA-204 have financial interests that could create a conflict of interest regulates Runx2 protein expression and mesenchymal pro- with regard to this work. genitor cell differentiation. Stem Cells 28, 357, 2010. GENE, PROTEIN, AND MICRORNA EXPRESSION IN CHONDRAL CELLS 227

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